Semi-preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of flavonoid glycosides from the leaves of Nelumbo nucifera (Lotus) by using a two-phase-solvent system composed of n-hexane–ethyl acetate–methanol–water (1:5:1:5, v/v/v/v). The targeted compounds isolated, collected and purified by HSCCC were analyzed by high performance liquid chromatography (HPLC). A total of 4.6mg of isoquercitrin, 9.1mg of hyperoside and 3.0mg of astragalin with the purity of 95.8%, 97.5% and 98.3%, respectively, were obtained in one-step separation and less than 6h from 80mg of crude extract from the leaves of N. nucifera. The chemical structures of all the three compounds were identified by MS, 1 H NMR, 13 C NMR. Astragalin was obtained from N. nucifera for the first time.